Identifying Candidate Genes Involved in the Regulation of Early Growth Using Full-Length Transcriptome and RNA-Seq Analyses of Frontal and Parietal Bones and Vertebral Bones in Bighead Carp (Hypophthalmichthys nobilis)

Abstract

Growth, one of the most important traits monitored in domestic animals, is essentially associated with bone development. To date, no large-scale transcriptome studies investigating bone development in bighead carp have been reported. In this study, we applied Isoform-sequencing technology to uncover the entire transcriptomic landscape of the bighead carp (Hypophthalmichthys nobilis) in early growth stage, and obtained 63,873 non-redundant transcripts, 20,907 long non-coding RNAs, and 1,579 transcription factors. A total of 381 alternative splicing events were seen in the frontal and parietal bones with another 784 events simultaneously observed in the vertebral bones. Coupling this to RNA sequencing (RNA-seq) data, we identified 27 differentially expressed unigenes (DEGs) in the frontal and parietal bones and 45 DEGs in the vertebral bones in the fast-growing group of fish, when compared to the slow-growing group of fish. Finally, 15 key pathways and 20 key DEGs were identified and found to be involved in regulation of early growth such as energy metabolism, immune function, and cytoskeleton function and important cellular pathways such as the arginine and proline metabolic pathway (p4ha1), FoxO signaling pathway (sgk1), cell adhesion molecules (b2m, ptprc, and mhcII), and peroxisome proliferator-activated receptor signaling pathway (scd). We established a novel full-length transcriptome resource and combined it with RNA-seq to elucidate the mechanism of genetic regulation of differential growth in bighead carp. The key DEGs identified in this study could fuel further studies investigating associations between growth and bone development and serve as a source of potential candidate genes for marker-assisted breeding programs.

Keywords: Iso-Seq; bighead carp; differential growth; differentially expressed unigene; full-length transcript.

FIGURE 1

GO classification of non-redundant transcripts in bone tissues of bighead carp.

FIGURE 2

Pathways associated with growth.

FIGURE 3

Venn graph of lncRNA transcripts from CPAT, CNCI, CPC, and Pfam analyses.

FIGURE 4

Distribution of GO classifications of DEGs in bone tissues of bighead carp. (A) Distribution of GO classifications of DEGs in the frontal and parietal bones. (B) Distribution of GO classifications of DEGs in the vertebral bones. Blue, green, and orange colors represented molecular function, cellular component, and biological process categories, respectively.

FIGURE 5

Pathway enrichment analysis of DEGs in bone tissues of bighead carp. (A) Enriched pathways from frontal and parietal bones between big and small groups of bighead carp. (B) Enriched pathways from vertebral bones between big and small groups of bighead carp.

FIGURE 6

Key genes related to early growth regulation in bone tissues of bighead carp. (A) Key genes related to early growth regulation in the frontal and parietal bones. (B) Key genes related to early growth regulation in the vertebral bones. The FPKM data of genes was used for heatmap construction. (C) The correlation network of key genes. The round nodes filled with diagonal lines indicate key genes related to early growth regulation in bone tissues. The round nodes filled with pure color indicate genes that interact with key genes. The interactions among different genes were represented by different colorful lines. Orange lines indicate predicted interactions. Purple lines indicate co-expression interactions. Gene abbreviations: pleckstrin homology domain-containing family J member 1 (plekhj1), SURP and G-patch domain-containing protein 1 (sugp1), prolyl 4-hydroxylase subunit alpha-1 (p4ha1), novel MHCII β chain protein (novel mhc II), protein tyrosine phosphatase type IVA (ptp4a), β2-microglobulin (b2m), serine/threonine-protein kinase Sgk1 (sgk1), receptor-type tyrosine-protein phosphatase C (ptprc), uncharacterized protein LOC101883013 (LOC101883013), myc target protein 1(myct1), golgi apparatus protein 1 (glg1), sodium/potassium-transporting ATPase subunit β (atp1b), stearoyl-CoA desaturase (scd), dopamine β-hydroxylase (dbh), succinate dehydrogenase [ubiquinone] cytochrome b small subunit B (sdhdb), MHC class II β precursor (mhcII), actin filament–associated protein 1-like 2 (afap1l2), acyl-CoA–binding protein (acbp), ubiquitin conjugation factor E4 B (ube4b), zinc finger protein 574 (znf574).

FIGURE 7

qRT-PCR validation of differently expressed genes in bone tissues of bighead carp from differential growth groups. (A) qRT-PCR validation of differently expressed genes of frontal and parietal bones between big and small groups of bighead carp. (B) qRT-PCR validation of differently expressed genes of vertebral bones between big and small groups of bighead carp. Gene expressions were expressed as mean normalized ratios (n = 3, ± SE). β-Actin was used as reference gene. Positive values denote up-regulation in big group compared to small group of bighead carp. Gene abbreviations: SURP and G-patch domain-containing protein 1 (sugp1), sodium/potassium-transporting ATPase subunit β (atp1b), receptor-type tyrosine-protein phosphatase C (ptprc), protein tyrosine phosphatase type IVA (ptp4a), stearoyl-CoA desaturase (scd), pleckstrin homology domain-containing family J member 1 (plekhj1), actin filament–associated protein 1-like 2 (afap1l2), microfibril-associated glycoprotein 4-like (msg4l), nuclear receptor co-activator 5 (ncoa5), golgi apparatus protein 1 (glg1), G-protein coupled receptor 126(gpr126), serine/threonine-protein kinase Sgk1(sgk1).

FIGURE 8

The layout plan of the study design. BG is the abbreviation of big bighead carp, and SG means small bighead carp.